The goal of this study is to develop a high throughput (HTS) assay to identify chemical compounds that induce or repress neural differentiation of human embryonic stem (hES) cells. The proposal is based on i) the need to identify agents that drive lineage specific differentiation in hES cells, ii) our ability to generate lineage specific reporter ES lines using BAC transgenesis, iii) improved protocols for the standardized growth of hEScells, iv) the availability of state-of-the-art robotic and screening tools for HTS implementation. This study should be a proof-of-principle application for the use of HTS assays to study early human development, provide novel insights into the pathways that control neural differentiation, and offer a prototype application for hES cell based HTS assays the will include future assays based on the use of our in-house generated siRNA library. The Specific Aims of the study are: 1. Generation of BAG transgenic human ES cell reporter lines for HTS assay (line WA-09) 2. Assay development to HTS format (line WA-09) 3. Biological validations and secondary screens of candidate compounds, (lines WA-09, WA-01, ES-03, ES-04. SA-01, SA-02, MI-01). The identification of novel compounds that direct neural differentiation of hES cells under defined in vitro conditions will also be an important contribution to translational stem cell efforts. Of particular importance will be the development of neural differentiation conditions that obviate the use of embryoid body formation or stromal mouse feeder co-culture protocols. Defined compound based neural differentiation may ultimately provide the most reproducible and safe differentiation strategy for future clinical applications.